U.S. patent application number 10/211412 was filed with the patent office on 2003-04-24 for gsk3 polypeptides.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Calderon-Cacia, Maria, Coit, Doris G., Fang, Eric Y., Hall, John A., Harrison, Stephen D., Medina-Selby, Angelica, Nguyen, Steve H., Zhong, Ziyang.
Application Number | 20030077798 10/211412 |
Document ID | / |
Family ID | 22826991 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077798 |
Kind Code |
A1 |
Harrison, Stephen D. ; et
al. |
April 24, 2003 |
GSK3 polypeptides
Abstract
The invention provides truncated GSK3 polypeptides capable of
crystallization, including GSK3.alpha. and GSK3.beta. polypeptides,
and use of these polypeptides to identify and optimize GSK3
inhibitors. Also provided are GSK3 polypeptides having at least one
substituted amino acid that differs from wild-type GSK3, wherein
the substituted amino acid is incapable of being phosphorylated.
The invention finds use in providing methods of identifying and
optimizing compounds useful for treating diseases mediated by GSK3
activity, including Alzheimer's disease, type 2 diabetes, and
inflammation.
Inventors: |
Harrison, Stephen D.;
(Albany, CA) ; Hall, John A.; (Rohnert Park,
CA) ; Calderon-Cacia, Maria; (Castro Valley, CA)
; Zhong, Ziyang; (Union City, CA) ; Fang, Eric
Y.; (Oakland, CA) ; Coit, Doris G.; (Petaluma,
CA) ; Nguyen, Steve H.; (San Francisco, CA) ;
Medina-Selby, Angelica; (San Francisco, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property R338
PO Box 8097
Emeryville
CA
98662-8097
US
|
Assignee: |
Chiron Corporation
Emeryville
CA
|
Family ID: |
22826991 |
Appl. No.: |
10/211412 |
Filed: |
July 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10211412 |
Jul 31, 2002 |
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09916109 |
Jul 25, 2001 |
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6465231 |
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60221242 |
Jul 27, 2000 |
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Current U.S.
Class: |
435/194 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
A61P 43/00 20180101; C12Q 1/485 20130101; A61P 3/10 20180101; A61P
29/00 20180101; C12Y 207/01037 20130101; G01N 2500/00 20130101;
A61P 25/28 20180101 |
Class at
Publication: |
435/194 ;
435/69.1; 435/320.1; 435/325; 536/23.2 |
International
Class: |
C12N 009/12; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
1. An isolated nucleic acid comprising a polynucleotide encoding a
polypeptide consisting essentially of SEQ ID NO:2, wherein said
polypeptide will crystallize and will have at least one biological
activity selected from the group consisting of (a) binding a GSK3
inhibitor; and (b) kinase activity.
2. An isolated nucleic acid comprising a polynucleotide encoding a
polypeptide consisting essentially of SEQ ID NO:3, wherein said
polypeptide will crystallize and will have at least one biological
activity selected from the group consisting of (a) binding a GSK3
inhibitor; and (b) kinase activity.
3. A vector comprising the polynucleotide of claim 1 or claim
2.
4. A polypeptide comprising between about 250 and 419 contiguous
amino acids of SEQ ID NO:1, wherein said polypeptide is
phosphorylated on tyrosine 216, said polypeptide will crystallize,
and said polypeptide will have at least one biological activity
selected from the group consisting of (a) binding a GSK3 inhibitor;
and (b) kinase activity.
5. A polypeptide consisting essentially of between about 278 and
419 contiguous amino acids of SEQ ID NO:1, wherein said polypeptide
exhibits at least 1% of the kinase activity of human
GSK3.beta..
6. A polypeptide consisting essentially of between about 285 and
384 contiguous amino acids of SEQ ID NO:1, wherein said polypeptide
exhibits at least 1% of the kinase activity of human
GSK3.beta..
7. A polypeptide consisting essentially of between about 351 and
384 contiguous amino acids of SEQ ID NO:1, wherein said polypeptide
exhibits at least 1% of the kinase activity of human
GSK3.beta..
8. A polypeptide consisting of the amino acid sequence of SEQ ID
NO:2.
9. A polypeptide consisting of the amino acid sequence of SEQ ID
NO:3.
10. An isolated nucleic acid comprising a polynucleotide encoding a
polypeptide consisting essentially of SEQ ID NO:5, wherein said
polypeptide will crystallize and will have at least one biological
activity selected from the group consisting of (a) binding to a
GSK3 inhibitor; and (b) kinase activity.
11. The nucleic acid of claim 10 wherein said polypeptide is
phosphorylated on tyrosine 279.
12. A vector comprising the polynucleotide of claim 10 or claim
11.
13. A polypeptide comprising between about 182 and 482 contiguous
amino acids of SEQ ID NO:4, wherein said polypeptide will
crystallize, and said polypeptide will have at least one biological
activity selected from the group consisting of (a) binding a GSK3
inhibitor; and (b) kinase activity.
14. A polypeptide consisting essentially of between about 182 and
386 contiguous amino acids of SEQ ID NO:4, wherein said polypeptide
exhibits at least 1% of the kinase activity of human
GSK3.alpha..
15. A polypeptide consisting essentially of between about 182 and
351 contiguous amino acids of SEQ ID NO:4, wherein said polypeptide
exhibits at least 1% of the kinase activity of human
GSK3.alpha..
16. A polypeptide consisting essentially of contiguous amino acids
S97 to S447 of SEQ ID NO: 1.
17. A polypeptide consisting essentially of the amino acid sequence
of SEQ ID NO:5.
18. A polynucleotide encoding a polypeptide consisting essentially
of SEQ ID NO:6.
19. A polypeptide consisting essentially of the amino acid sequence
of SEQ ID NO:6.
20. A polynucleotide encoding a polypeptide consisting essentially
of SEQ ID NO:7.
21. A polypeptide consisting essentially of the amino acid sequence
of SEQ ID NO:7.
22. A polynucleotide encoding a non-phosphorylated human GSK3
polypeptide, wherein said non-phosphorylated polypeptide differs
from native GSK3 in at least one and not more than ten amino
acids.
23. The polynucleotide of claim 22 wherein tyrosine at position 216
of SEQ ID NO:1 is substituted for by a non-phosphorylatable amino
acid.
24. The polynucleotide of claim 23 wherein said
non-phosphorylatable amino acid is phenylalanine.
25. The polynucleotide of claim 22 wherein tyrosine at position 279
of SEQ ID NO:4 is substituted for by a non-phosphorylatable amino
acid.
26. The polynucleotide of claim 25 wherein said
non-phosphorylatable amino acid is phenylalanine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/221,242 filed Jul. 27, 2000, where this
provisional application is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention provides materials and methods relating to
identification and optimization of selective inhibitors of glycogen
synthase kinase 3 (GSK3), and also relates to methods of treating a
condition mediated by GSK3 activity. Such conditions include
Alzheimer's disease, type 2 diabetes, and inflammation.
[0004] 2. Description of the Related Art
[0005] Glycogen synthase kinase 3 (GSK3) is a proline-directed
serine/threonine kinase originally identified as an activity that
phosphorylates glycogen synthase as described in Woodgett, Trends
Biochem Sci 16:177-181 (1991). The role in glucose metabolism has
been elaborated recently in Summers et al., J Biol. Chem.
274:17934-17940 (1999). GSK3 consists of two isoforms, .alpha. and
.beta., and is constitutively active in resting cells, inhibiting
glycogen synthase by direct phosphorylation. Upon insulin
activation, GSK3 is inactivated, thereby allowing the activation of
glycogen synthase and possibly other insulin-dependent events. GSK3
is inactivated by other growth factors or hormones that, like
insulin, signal through receptor tyrosine kinases. Examples of such
signaling molecules include IGF-1 and EGF as described in Saito et
al., Biochem. J. 303:27-31 (1994), Welsh et al., Biochem. J.
294:625-629 (1993), and Cross et al., Biochem. J. 303:21-26 (1994).
GSK3 has been shown to phosphorylate .beta.-catenin as described in
Peifer et al., Develop. Biol. 166:543-56 (1994). Other activities
of GSK3 in a biological context include GSK3's ability to
phosphorylate tau protein in vitro as described in Mandelkow and
Mandelkow, Trends in Biochem. Sci. 18:480-83 (1993), Mulot et al.,
Febs Lett 349: 359-64 (1994), and Lovestone et al., Curr. Biol.
4:1077-86 (1995), and in tissue culture cells as described in
Latimer et al., Febs Left 365:42-6 (1995). Selective inhibition of
GSK3/may be useful to treat or inhibit disorders mediated by GSK3
activity.
[0006] There is a need in the art for compositions and molecules
that bind to or interact with GSK3, thereby mediating GSK3
activity. The invention meets this need by providing crystallizable
GSK3 polypeptides useful for design and optimization of GSK3
inhibitors.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides GSK3.beta. molecules with N- and
C-terminal truncations, wherein the molecules are capable of
crystallization.
[0008] The invention further provides GSK3.beta. molecules
truncated at amino acid R.sup.344, R.sup.354, T.sup.364, A.sup.374,
and I.sup.384.
[0009] The invention provides a polypeptide consisting essentially
of SEQ ID NO:2 or SEQ ID NO:3, polynucleotides encoding these
polypeptides, and vectors comprising these polynucleotides.
[0010] The invention still further provides GSK3.beta. molecules
wherein translation of the molecule begins at G.sup.34, T.sup.39,
P.sup.44, D.sup.49 or V.sup.54.
[0011] The invention also provides GSK3.alpha. molecules with N-
and C-terminal truncations, wherein the molecules are capable of
crystallization.
[0012] The invention further provides a GSK3.alpha. molecule
wherein translation of the molecule begins at S.sup.97 and ends at
S.sup.447, polynucleotides encoding this polypeptide, and vectors
comprising these polynucleotides.
[0013] The invention further provides a method of identifying a
GSK3 polypeptide capable of crystallization, comprising: (a)
providing a truncated GSK3 polypeptide; (b) testing the polypeptide
for formation of crystals.
[0014] The invention also provides GSK3 polypeptides capable of
interacting with inhibitors of GSK3.
[0015] The invention further provides a method of identifying an
enzymatically active GSK3 polypeptide, comprising: (a) providing a
truncated GSK3 polypeptide; (b) contacting the polypeptide with a
substrate of GSK3; and (c) measuring the kinase activity of the
polypeptide after contacting the polypeptide with the substrate,
wherein the polypeptide is active if it shows >0.01.times.the
activity of the full-length enzyme and preferably >0.1.times.the
activity of the full-length enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS
[0016] FIG. 1 provides the polypeptide sequence of human GSK3.beta.
(SEQ ID NO:1).
[0017] FIG. 2 provides the polypeptide sequence of truncated
GSK3.beta. polypeptide 557 (SEQ ID NO:2). The first ten amino acids
represent a Glu-tag, followed by a Gly linker before Met at
position 1.
[0018] FIG. 3 provides the polypeptide sequence of truncated
GSK3.beta. polypeptide 580 (SEQ ID NO:3). The first ten amino acids
represent a Glu-tag, followed by a Gly linker before Gly at
position 34.
[0019] FIG. 4 provides the polypeptide sequence of human
GSK3.alpha. (SEQ ID NO:4).
[0020] FIG. 5 provides the polypeptide sequence of human
GSK3.alpha. truncated at position 447 (SEQ ID NO:5).
[0021] FIG. 6 provides the polypeptide sequence of human
GSK3.alpha. truncated at position 97 (SEQ ID NO:6).
[0022] FIG. 7 provides the polypeptide sequence of human
GSK3.alpha. from position 97 to position 447 (SEQ ID NO:7).
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides materials and methods for identifying
and optimizing inhibitors of GSK3, including GSK3.alpha. and
GSK3.beta.. The provided materials include C- and N-terminal
truncated GSK3.beta. molecules that are capable of crystallization
and may, but need not, retain GSK3 kinase activity, preferably more
than 0.01.times.the activity of the full-length enzyme and more
preferably more than 0.1.times.the activity of the full-length
enzyme. There is a need in the art for such inhibitors, in view of
the role of GSK3 in a variety of diseases and conditions, including
Alzheimer's disease, type 2 diabetes and inflammation. Such
inhibitors can be identified, and identified inhibitors can be
optimized, using the crystallizable GSK3 polypeptides of the
invention.
[0024] The invention provides a variety of GSK3.beta. polypeptides
that differ from the native polypeptide at the C- and/or
N-terminus. The amino acid sequence of GSK3.beta. is shown in FIG.
1 (SEQ ID NO: 1). Included within the scope of the invention are
any and all truncations of GSK3.beta. polypeptide wherein the
truncated polypeptide is capable of crystallization and may, but
need not, retain kinase activity as measured using the kinase
assays described herein. Persons of skill in the art will realize
that limited mutation of the protein, or certain post-translational
modifications, might be sufficient to inactivate the kinase yet
retain the essential 3D structure. Such inactive but structurally
related molecules would also be useful for the design and
optimization of inhibitors. Kinase assays are disclosed in U.S.
Pat. Nos. 6,057,117 and 6,057,286, which are incorporated herein by
reference. The percent activity that is retained, if any, is not
crucial. Methods of assaying activity in the presence and absence
of an inhibitor are described herein.
[0025] The invention provides numerous truncated GSK3.beta.
polypeptides that meet these criteria. A preferred polypeptide is
designated BV557 in which the C-terminal amino acid is R.sup.384.
This molecule has been successfully crystallized. Additional active
polypeptides include those with truncations at amino acid
R.sup.344, R.sup.354, A.sup.374, and I.sup.384.
[0026] The invention also provides truncated GSK3.alpha.
polypeptides, including a GSK3.alpha. polypeptide beginning at
S.sup.97 and ending at S.sup.447.
[0027] Additional truncated GSK3 polypeptide include those
beginning with an N-terminal amino acid that differs from that of
the native protein in that 1 or more amino acids are deleted from
the N-terminus. Preferred N-terminal truncations include GSK3.beta.
molecules wherein translation of the molecule begins at G.sup.34,
T.sup.39, P.sup.44, D.sup.49 or V.sup.54. An example is BV580
(amino acids 34 to 384) which has been crystallized.
[0028] The invention is not limited to these disclosed truncated
molecules. Using the methods and assays described herein, one of
skill can construct additional truncated molecules, such as those
having 36-76 amino acids deleted from the C-terminus, and/or 35-54
amino acids deleted from the N-terminus. Such deletions can occur
individually, or a polypeptide can have both an N-terminal deletion
and a C-terminal deletion. It is preferable but not necessary that
the kinase domain remain relatively intact as reflected by the
detection of enzymatic activity, such as by using the assays
described herein. It is also desirable, although not essential,
that the enzymatic activity be capable of inhibition by a known
GSK3 inhibitor, such as lithium. A truncated molecule meeting these
criteria will be suitable for testing GSK3 inhibitors as potential
therapeutic agents, and for optimizing GSK3 inhibitors.
[0029] A truncated GSK3.beta. polypeptide of the invention can
consist of between about 250 and 419 contiguous amino acids of SEQ
ID NO: 1; preferably between about 278 and 419 contiguous amino
acids of SEQ ID NO: 1; more preferably between about 285 and 384
contiguous amino acids of SEQ ID NO: 1; and most preferably between
about 351 and 384 contiguous amino acids of SEQ ID NO:1. Preferred
truncated GSK313 polypeptides include those beginning at amino acid
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, or 62 of SEQ ID NO:1, and ending at
amino acid 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,
351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,
364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,
377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,
390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,
403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,
416, 417, 418 or 419 of SEQ ID NO:1. The polypeptide can begin with
any one of the listed beginning amino acids and end with any one of
the ending amino acids. Exemplary and non-limiting embodiments
begin at amino acid 34, 39, 44 or 54 and end at amino acid 420.
Other particularly preferred embodiments begin at about amino acid
1 and end at amino acid 340, 344, 354, 374, 384, 385, 386, 387,
388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,
401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,
414, 415, 416, 417, 418, 419, or 420.
[0030] The truncated GSK3.alpha. polypeptide of the invention can
consist of between about 182 and 482 contiguous amino acids of SEQ
ID NO: 4, preferably between about 182 and 386 contiguous amino
acids of SEQ ID NO:4, more preferably between about 182 and 351
contiguous amino acids of SEQ ID NO:4, and most preferably from
about S.sup.97 to S.sup.447 of SEQ ID NO:4.
[0031] The truncated GSK3 polypeptides can be prepared by any
method known in the art. One method involves expression of a
suitably prepared polynucleotide encoding a polypeptide having the
desired truncation. For example, a preferred polypeptide of the
invention, BV557, was prepared by creating a construct encoding
GSK3.beta. starting at M.sup.I and ending at I.sup.384, as
described in the Examples. Briefly, insect cells were transfected
with baculovirus vector (designated pBlueBac4.5.Glu.GSK3B.DC.-
1384#28), which encodes BU557, and the protein was extracted from
the lysed cells. The protein was purified by affinity
chromatography using an anti glu-tag monoclonal antibody
immobilized on a Sepharose column. Activity of the purified protein
was assayed using the in vitro kinase assay described in U.S. Pat.
No. 6,057,286.
[0032] The Examples herein describe the production of BV557, BV580,
and other truncated GSK3 polypeptides by expression of vectors
encoding the polypeptides, followed by isolation and purification
of the polypeptides. The polypeptide can also be produced by
enzymatic cleavage of a native GSK3 protein, using methods known in
the art. Other suitable methods include expression of a
polynucleotide encoding a truncated polypeptide in a variety of
cell types, including mammalian, bacterial, or yeast cells.
However, the preferred cell for expression of the polypeptide is an
insect cell, preferably a baculovirus-infectable insect cell, such
as a Sf9 cell.
[0033] The invention also provides unphosphorylated forms of GSK3
wherein the ATP binding site is identical to that of the wild-type
protein. Such forms include Y216 non-phosphorylated GSK3.beta. and
Y279 non-phosphorylated GSK3.alpha.. Other forms include constructs
with at least one amino acid change that prevents phosphorylation,
such as GSK3.beta. in which Y216 is changed to F216, and
GSK3.alpha. in which Y279 is changed to F279. These forms are
suitable for inhibitor binding assays to identify inhibitors of
GSK3. The invention provides a GSK3.beta. molecule in which
position 216 is not phosphorylated. We have demonstrated that a
GSK3.beta. peptide with Y216 mutated to F216 crystalized and
exhibits a structure in which the ATP-binding site is not
substantially different from the un-mutated peptide.
[0034] Additional single and multiple amino acid changes include
S.sup.9 to A.sup.9 in GSK3.beta. and S.sup.21 to A.sup.21 in
GSK3.alpha..
[0035] These changes in phosphorylation, or ability to be
phosphorylated, are optionally incorporated into the truncated
forms of GSK3.alpha. and GSK3.beta. disclosed herein.
[0036] The invention therefore provides GSK3 molecules suitable for
design and optimization of inhibitors of GSK3 as pharmaceutical
agents.
[0037] The GSK3 constructs of the invention are capable of
crystallization. In purified form the constructs bind to inhibitors
in a manner that is comparable to inhibitor binding to the native
GSK3 polypeptide, due to the retention of the correct folding
conformation at the inhibitor binding site. Potential to
crystallize is measured using a variety of assays including
specific activity, aggregation, microheterogeneity. (See, for
example, Table 1). These parameters are indicative of the purity of
the preparation and of the solubility of the construct. The
specific activity is also a preferred assay for detecting binding
of an inhibitor to the correct binding site of the GSK3 construct.
Another suitable method is fluorescence polarization. Briefly, a
putative inhibitor, with an attached fluorophore, tumbles freely in
solution. Thus when the fluorophore is excited by polarized light,
the emitted light which is produced after a finite delay now has
random polarity and the emitted light is no longer polarized. In
the presence of a GSK3 construct with an intact inhibitor binding
site, the tumbling rate is slowed sufficiently to ensure that, even
though the light emission is delayed with respect to the
excitation, the fluorophore has only moved very slightly. Thus, the
excited light maintains polarization. A measurement of fluorescence
polarization therefore indicates whether or not the GSK3 construct
is suitable for identifying and optimizing an inhibitor. The
fluorophore can be attached to a compound such as staurosporine
(ICN Pharmaceuticals, Inc., Costa Mesa, Calif.). GSK3 constructs
may not retain kinase activity, but their inhibitor binding can
still be assessed using fluorescence polarization assays.
[0038] The term "truncated glycogen synthase kinase 3" or
"truncated GSK3" as used herein refers to GSK3.alpha. or
GSK3.beta.. GSK3 is a protein originally identified by its
phosphorylation of glycogen synthase as described in Woodgett et
al, Trends Biochem Sci, 16:177-181(1991). Synonyms of GSK3 are tau
protein kinase I (TPK I), FA kinase and kinase FA. Mammalian forms
of GSK3 have been cloned as described in Woodgett, EMBO J.
9(8):2431-2438 (1990). Inhibitors of truncated GSK3 polypeptides
can be inhibitors of any of the known forms of GSK3, including
either GSK3.alpha. or GSK3.beta. or both. Truncated polypeptides of
the invention possess one or more of the bioactivities of the GSK3
protein, including kinase activities such as polymerizing tau
protein, or phosphorylating glycogen synthase, for example. Thus,
truncated GSK3 polypeptides useful for designing and optimizing
inhibitors of GSK3 can have sequence identity of at least 40%,
preferably 50%, preferably 60%, preferably 70%, more preferably
80%, and most preferably 90% to the amino acid sequence of the
native protein, wherever derived, from human or nonhuman sources.
The polynucleotides encoding a GSK3 polypeptide can have 60%,
preferably 70%, more preferably 80%, more preferably 90% and most
preferably 95% sequence identity to a native polynucleotide
sequence of GSK3. Also included, therefore, are alleles and
variants of the native polynucleotide sequence such that the
polynucleotide encodes an amino acid sequence with substitutions,
deletions, or insertions, as compared to the native sequence.
[0039] The term "peptide substrate" refers to a peptide or a
polypeptide or a synthetic peptide derivative that can be
phosphorylated by GSK3 activity in the presence of an appropriate
amount of ATP or a phosphate donor. Detection of the phosphorylated
substrate is generally accomplished by the addition of a labeled
phosphate that can be detected by some means common in the art of
labeling, such as radiolabeled phosphate. The peptide substrate may
be a peptide that resides in a molecule as a part of a larger
polypeptide, or may be an isolated peptide designed for
phosphorylation by GSK3.
[0040] As disclosed in U.S. Pat. Nos. 6,057,117 and 6,057,286, in
vitro methods of assaying GSK3 activity include constructing
peptide substrates. The peptide substrate can be any peptide
substrate phosphorylatable by GSK3, and may be a peptide substrate
including the formula: anchor ligand-(X).sub.nSXXXS(X).sub.m (SEQ
ID NO:8) (wherein X is any amino acid, n is any integer, m is any
integer, and preferably n+m+5<20, i.e. n+m<15)
prephosphorylated at the C terminal serine. The assay is performed
by contacting the substrate with truncated GSK3 polypeptide in the
presence of phate-ATP, a substrate anchor, and optionally a
candidate inhibitor. The identifying an inhibitor of GSK3 kinase
activity includes contacting a oupled to an anchor ligand with
truncated GSK3 polypeptide in the abeled .gamma.phosphate-ATP, a
substrate anchor, and candidate inhibitor, measuring an
incorporation of radiolabel into the peptide substrate, then, in a
separate assay vessel contacting a peptide substrate coupled to an
anchor ligand with truncated GSK3 in the presence of radiolabeled
.gamma.phosphate-ATP, and a substrate anchor, and measuring
incorporation of radiolabel into said peptide substrate; ultimately
an inhibitor of truncated GSK3 kinase activity is identified by a
reduction of label incorporation in the assay with the candidate
inhibitor as compared to the assay without the candidate
inhibitor.
[0041] To conduct the in vitro kinase assay of the invention using
microwells, scintillant may be present by pre-coating the wells
with a scintillant material, or by adding it later following a wash
step, as described in Example 4. The scintillant can be obtained
from Packard, Meridian, Conn. Wells coated with scintillant are
then in addition coated with streptavidin as a substrate anchor,
where biotin is the anchor ligand on the peptide. Alternatively,
the streptavidin can be present on agarose beads containing
scintillant or may be coated on an otherwise untreated plate to
which scintillant is added subsequently. In any event, the
streptavidin in the wells binds the biotin that contacts it.
Following an assay using radiolabeled ATP, the radiolabel
incorporated into the phosphorylated substrate that has been
conjugated to the biotin will cause the scintillant to emit light.
Where the streptavidin is attached to agarose beads containing
scintillant, binding a biotin-conjugated radiolabeled peptide
substrate will cause the beads to scintillate. In both the case of
the wells lined with the scintillant, and the agarose beads
containing scintillant, a reduction in scintillation as compared to
a control amount of scintillation measured under non-inhibitory
conditions, indicates the presence of a functional inhibitor of
GSK3 activity. If the peptide has been phosphorylated by GSK3 with
.sup.32P-labeled or .sup.33P-labeled phosphate, radioactive decay
will cause the scintillant present in a microwell or mixed in
agarose beads that are present in the reaction mixture to emit
light and the measure of the amount of light emitted will be a
measure of the activity of GSK3 in the assay. Low activity of GSK3
observed in the presence of a candidate inhibitor, as compared to
the activity of GSK3 in the absence of the inhibitor, may indicate
that the inhibitor is functional and can inhibit GSK3 kinase
activity. In any case, an excess of streptavidin over peptide
should be loaded into each well or should be affixed to the agarose
beads.
[0042] GSK3 inhibitory activity can be measured using a cell-free
assay as disclosed in publication WO 99/65897, and as described in
Example 4 herein. Activity can also be measured using a cell-based
assay. Briefly, a cell line, such as a Cos cell line, is
transfected with Tau and with a GSK3 polypeptide. The
phosphorylation of Tau at a specific site is monitored using a
monoclonal antibody, as phosphorylation at that site is dependent
on GSK3 activity.
[0043] Exemplary polypeptides of the invention include the
following truncated polypeptides with reference to SEQ ID NO:
1:
[0044] GSK3.beta. truncated at R.sup.344
[0045] GSK3.beta. truncated at R.sup.354
[0046] GSK3.beta. truncated at T.sup.364
[0047] GSK3.beta. truncated at A.sup.374
[0048] GSK3.beta. truncated at I.sup.384
[0049] GSK3.beta. beginning at G.sup.34
[0050] GSK3.beta. beginning at T.sup.39
[0051] GSK3.beta. beginning at P.sup.44
[0052] GSK3.beta. beginning at D.sup.49
[0053] GSK3.beta. beginning at V.sup.54
[0054] The above truncations can be combined, providing a
GSK3.beta. polypeptide beginning at any of G.sup.34, T.sup.39,
P.sup.44, D.sup.49, or V.sup.54, and ending at any of R.sup.344,
R.sup.354, T.sup.364, A.sup.374, or I.sup.384.
[0055] Other exemplary polypeptides of the invention include the
following truncated polypeptides with reference to SEQ ID NO:4:
[0056] GSK3.alpha. truncated at S.sup.447.
[0057] GSK3.alpha. beginning at S.sup.97.
[0058] GSK3.alpha. beginning at S.sup.97 and truncated at
S.sup.447.
[0059] A truncated GSK3 polypeptide of the invention can be
selected on the basis of one or more parameters. A polypeptide will
preferably crystallize in a form that is similar to that of native
GSK3, with correct folding at and around the inhibitor binding
site. Crystallization can be performed using a Crystal Screen Kit
(Hampton Research, Laguna Niguel, Calif.), or methods described by
Jancarik, J. et al., J. Appl. Cryst. 24:409-411, 1991. The
potential of a polypeptide to form crystals can be evaluated on the
basis of specific activity, purity, homogeneity, mass spectrometry,
aggregation, and dynamic light scattering. A preferred truncated
polypeptide will meet the following parameters: purity of at least
90%; less than 100% aggregation at 4.degree. C. at two weeks; and
less than 50% heterogeneity (50% or greater of the desired form). A
most preferred truncated polypeptide will have a purity of at least
98%, no aggregation at 4.degree. C. at two weeks; and less than 5%
heterogeneity (unphosphorylated form). Such parameters indicate
that the polypeptide preparation is likely to crystallize, making
it suitable for discovering and optimizing GSK3 inhibitors.
[0060] A prerequisite for crystallization is to obtain a
sufficiently concentrated stock of protein. Not all GSK3 constructs
will remain soluble at the required concentration. A preferred
concentration is >1 mg/ml, more preferred is >5 mg/ml, and
most preferred is >10 mg/ml.
[0061] The polypeptides disclosed herein as 557 (SEQ ID NO:2), 580
(SEQ ID NO:3), 458, and 524 meet the criteria described above (see
Example 3). Polypeptide 458 consists of amino acids 1-420 of SEQ ID
NO:1 plus the following addition at the N-terminus: EFMPTEAMAAPKRVI
(SEQ ID NO:8). Polypeptide 524 consists of amino acids 1-420 of SEQ
ID NO:1 plus the following addition at the N-terminus: EYMPMEGGG
(SEQ ID NO:9). Other modified or truncated GSK3 polypeptides can be
prepared and tested as described herein.
EXAMPLES
[0062] The following examples are exemplary only, and are not
intended to limit the invention.
Example 1
Preparation and Purification of GSK3.beta. Construct 557
[0063] Lysis and Extraction. Insect cell slurry from Sf9 cells
(about 10 g) from a 1 liter flask growth was combined with 30 ml of
lysate buffer: 20 mM Tris, pH 8.0/80 mM NaCl/1 mM MgCl.sub.2/1 mM
Arsenate/1 mM Tungstenate/1 mM PMSF/0.5 mg Leupeptin/0.2 mg
Aprotinin. Cells were lysed using a Dounce homogenizer. Improved
extraction of the protein was accomplished by the addition of 5%
glycerol and 0.2% octylglucoside. The mixture was allowed to stir,
on ice, for 30 minutes. The total lysate was centrifuged at
39000.times.g for 25 minutes at 4.degree. C. The resulting
supernatant contained the extracted GSK3-.beta. #557.
[0064] Ion Exchange Chromatography. The following materials and
conditions were used: The resin was Fractogel EMD SO.sub.3-- (M);
the column diameter was 1.6 cm and the column volume was 10 ml. The
column was run at a flowrate of 90 cm/hour using equilibration
buffer of 20 mM Na Phosphate/5% Glycerol, pH 7.5. Chromatography
was carried out at 4.degree. C.
[0065] The lysate supernatant was diluted 1:1 with S-fractogel
equilibration buffer, and loaded onto the equilibrated column. The
column was washed with a total of 14 column volumes of
equilibration buffer. The GSK3-.beta. was eluted with a linear salt
gradient, over 20 column volumes, to equilibration buffer plus 1M
NaCl. 3 ml/fraction was collected during gradient elution. The pool
was made based on SDS-PAGE and Western blot results of the
fractions collected. Fractions 13-24 were pooled.
[0066] Affinity Chromatography was performed using the following
materials and procedures: The resin was anti glu-tag monoclonal
antibody immobilized onto Protein G Sepharose, and the
equilibration buffer was PBS/0.3M NaCl/0.2% octylglucoside/10%
Glycerol. The column diameter was 1.6 cm and the column volume was
13 ml. The flow rate was 30 cm/hour during load and wash, and 15
cm/hour during elution.
[0067] The S-Fractogel pool was loaded at 30 cm/hour onto
equilibrated column. The column was washed down to absorbance
baseline with approximately 6 column volumes of equilibration
buffer, and GSK3.beta. was eluted with 50 ml of equilibration
buffer containing 2 mg of elution peptide (EYMPTD). The flow rate
during elution was lowered to 15 cm/hour. 2 ml/fractions were
collected during the elution. Based on SDS-PAGE results, elution
fractions 6-17 were pooled with a total volume of 24 ml.
[0068] Final Yield. The affinity column pool, at a concentration of
0.17 mg/ml, contained 4.1 mg of GSK3.beta. #557. This translates to
a final yield of 4.1 mg purified 557/liter of growth. Purity, after
this 2 column purification, was estimated at >95% by visual
inspection of SDS-PAGE results.
Example 2
Preparation and Purification of GSK3 Construct 580
[0069] Extraction. SF9 cell paste from a 10 L fermentation was
washed with 100 mL PBS (10 mM NaPi, pH 7.5, 150 mM NaCl) and then
resuspended with 300 mL of Buffer H (20 mM Tris, pH 7.5, 1 mM
Tungstate, 1 mM Arsenate, 5 mM DTT, 10 .mu.g/mL Leupeptin, 1
.mu.g/mL pepstatin A, 10% glycerol, 0.35% Octyl glucoside, 1 mM
Mg.sup.2+). Cells were homogenized in a 100-mL Dounce Homogenizer
(20 strokes with pestle B). The combined homogenate was centrifuged
in a Ti45 rotor at 40,000 rpm for 35 minutes to remove cell debris
and nuclei. The supernatant from the centrifugation were carefully
decanted and filtered through 0.45 .mu. filter.
[0070] S-Fractogel. 100 mL S-Fractogel (EM Science, Cat #18882) was
packed into a 3.2 cm.times.12.5 cm column and equilibrated with
>1 L of buffer A (20 mM Tris, pH 7.5, 10% glycerol). The
filtrate from the previous step was loaded at 15 mL/min onto the
column. The column was washed with 1 L of buffer A and then eluted
with a linear gradient from 0 to 1 M NaCl in buffer A over 20
column volumes. The eluant was fractionated into 20 mL each.
Fractions containing GSK3 were detected by Western Blot using
anti-GSK antibody (Santa Cruz Biotech, Cat # SC-7291). The
Western-Blot positive fractions were pooled and mixed with equal
volume of buffer M (20 mM Tris, pH 7.5, 10% glycerol, 3.1 M NaCl)
and filtered through a 0.45.mu. filter. The filtrate was saved for
Phenyl-650 M chromatography.
[0071] Phenyl-650 M. 37.5 mL Phenyl-650 M (Tosohass, Cat # 014943)
was packed into a 2.2.times.10 cm column and equilibrated with 500
mL of buffer C (20 mM Tris, pH 7.5, 10% glycerol, 1.6 M NaCl).
Filtrate from S-fractogel step was loaded onto the column at 7.5
mL/min. After the loading was completed, the column was washed with
6.5 cv buffer C and eluted with a linear gradient from 0% to 100%
Buffer D (20 mM Tris, pH 7.5, 10% glycerol) over 20 column volumes.
Fractions were collected at 15 mL each and GSK containing fractions
were detected by Western Blot using anti-GSK antibody. The Western
positive fractions were pooled and loaded onto a Glu-tag antibody
affinity column.
[0072] Glu-tag antibody Affinity Chromatography. Use of a Glu-tag
is described in Rubinfeld et al., Cell 65:1033-1042, 1991, and a
hybridoma expressing anti-Glu-tag antibody is described in
Grussenmyer et al., PNAS 82:7952-7954 (1985). 50 mg of the Glu-tag
antibody was immobilized onto 25 mL of Affi-Gel 10 (BioRAD, Cat
#153-6046) and packed into a 2.2.times.6.5 cm column. The column
was equilibrated with 200 mL of buffer E (20 mM Tris, pH 7.5, 10%
glycerol, 0.3 M NaCl, 0.2% Octylglucoside) and the fraction pool
from the Phenyl-650 M step was loaded at 1.0 mL/min. After the
loading was completed, the column was washed with 100 mL of buffer
E and then eluted with 60 mL Glu-tag peptide (100 .mu.g/mL) in
Buffer E and fractionated into 5 mL each. GSK containing fractions
were detected with SDS-PAGE and Coomassie Blue staining. These
fractions were pooled and concentrated to approximately 6 mg/mL in
an Amicon concentrator using a 10 k MWCO YM10 membrane. The
concentrated material was then ready for crystallization.
Example 3
Activity of Truncated GSK3.beta. Polypeptides
[0073] A reaction mixture was prepared containing 5.9 .mu.M
prephosphorylated SGSG-linked CREB peptide (Wang et al., Anal.
Biochem., 220:397-402 (1994)).mu. in reaction buffer (5 mM Tris, pH
7.5, 5 mM DTT; 1 mM MgCl2, 0.01% BSA) containing the desired amount
of truncated GSK3 polypeptide. ATP was added (specific activity 5.3
Ci/mmol) to 25 .mu.M final concentration and the mixture was
incubated for 20 min. at room temperature. The reaction was stopped
by transferring 30 .mu.l onto a P81 filter disc (Whatman). The disc
was washed four times in 150 ml of 75 mM H.sub.3PO.sub.4 for 5
minute each. The filter was air dried and counted under 5 ml
scintillation fluid. The specific activity was counted by
determining the ratio of counts (in cpm) by the mass of GSK3 in the
reaction (in .mu.g).
[0074] The specific activity for construct 557 was
4.3.times.10.sup.7 cpm/.mu.g; for construct 458, 2.8.times.10.sup.7
cpm/.mu.g; and for construct 524, 2.2.times.10.sup.7 cpm/.mu.g.
1TABLE 1 Mean Specific Activity Concentration Aggregation
Aggregation Heterogeneity Construct Purity cpm/.mu.g N mg/ml at 4
Degrees at RT % 458 >98% 2.8 .times. 10.sup.7 35 11.5 11% @ >
2 overnight 10-20% weeks unphosphorylated 557 >98% 4.3 .times.
10.sup.7 7 12.7 none @ > 2 overnight 5% weeks unphosphorylated
524 >98% 2.2 .times. 10.sup.7 24 10 ND <5% unphosphorylated N
= number of assays used to determine "mean specific activity."
Example 4
Screening for GSK3 Inhibitory Activity Using a Cell-Free Assay
[0075] Compounds to be tested as GSK3 inhibitors are dissolved in
DMSO, then tested for inhibition of human GSK3.beta.. Expression of
GSK3.beta. is described, for example, in Hughes et al., Eur. J.
Biochem., 203:305-11 (1992), which is incorporated herein by
reference. An aliquot of 300 .mu.l of substrate buffer (30 mM
tris-HCl, 10 mM MgCl.sub.2, 2 mM DTT, 3 .mu.g/ml GSK3.beta.) and
0.5 .mu.M biotinylated prephosphorylated SGSG-linked CREB peptide
(Chiron Technologies PTY Ltd., Clayton, Australia) is dispensed
into wells of a 96 well polypropylene microtiter plate. 3.5
.mu.l/well of DMSO containing varying concentrations of each
compound to be assayed or staurosporine (a known kinase inhibitor
used as a positive control, or a negative control) (i.e., DMSO
only), is added and mixed thoroughly. The reactions is then
initiated by adding 50 .mu.l/well of 1 .mu.M unlabeled ATP and
1-2.times.10.sup.7 cpm .gamma..sup.33P-labeled ATP, and the
reaction is allowed to proceed for about three hours at room
temperature.
[0076] While the reaction is proceeding, streptavidin-coated
Labsystems "Combiplate 8" capture plates (Labsystems, Helsinki,
Finland) are blocked by incubating them with 300 .mu.l/well of PBS
containing 1% bovine serum albumin for at least one hour at room
temperature. The blocking solution is then removed by aspiration,
and the capture plates are filled with 100 .mu.l/well of stopping
reagent (50 .mu.M ATP/20 mM EDTA).
[0077] When the three hour enzyme reaction is finished, triplicate
100 .mu.l aliquots of each reaction mix are transferred to three
wells containing stopping solution, one well on each of the three
capture plates, and the well contents are mixed well. After one
hour at room temperature, the wells of the capture plates are
emptied by aspiration and washed five times using PBS and a 12
channel Corning 430474 ELISA plate washer. Finally, 200 .mu.l of
Microscint-20 scintillation fluid is added to each well of the
plate. The plates are coated with plate sealers, then left on a
shaker for 30 minutes. Each capture plate is counted in a Packard
TopCount scintillation counter (Meridian, Conn.) and the results
are plotted as a function of compound concentration.
[0078] Compounds identified using this method can be further
optimized by testing their ability to bind to truncated GSK3
polypeptides of the invention, using the fluorescence polarization
assay, for example, for truncated polypeptides that do not exhibit
GSK3 kinase activity. Alternatively, a truncated GSK3 polypeptide
of the invention can be used in place of the native GSK3
protein.
Example 5
Screening for Inhibition of Tau Protein Phosphorylation
[0079] A. Transient Transfection of COS Cells with Expression
Plasmid Encoding Truncated GSK3 and Tau Expression Plasmid
Construction
[0080] COS cells are maintained in T25 tissue culture flasks in
high glucose MEM medium/5% fetal bovine serum. Cells from a
confluent T25 flask are harvested and 80,000 cells/well are seeded
into Corning 6-well tissue culture plates in a final volume of 2
ml/well of medium. The cells are left to grow at 37.degree. C. for
48 hours. The cells are then washed twice in Opti-MEM containing no
fetal bovine serum, and finally the cells are left in 1 ml of
Opti-MEM.
[0081] Polynucleotide encoding tau protein is subcloned into
plasmid pSG5 under an early SV40 promoter to generate a tau
expression plasmid. The cloning of cDNA encoding tau protein is
generally described in Goedert et al., EMBO Journal, 8(2):393-399
(1989), which is incorporated herein by reference. A GSK3
expression plasmid is prepared by subcloning polynucleotide
encoding truncated GSK3 into pCG, which is an ApEVRF derivative
described in Giese et al., Genes & Development, 9:995-1008
(1995) and Matthias et al., Nucleic Acid Research, 17:6418 (1989),
both of which are incorporated herein by reference. The
polynucleotide can encode any of the truncated GSK3 polypeptides of
the invention.
[0082] The following solutions are prepared in 1.5 ml Eppendorf
tubes:
[0083] Solution A: for each transfection, 2 .mu.g of DNA (tau
expression plasmid) and 0.7 .mu.g of DNA (GSK3 expression plasmid)
are diluted into 100 .mu.l of Opti-MEM (Gibco BRL); Solution B: for
each transfection, 8 .mu.l of Lipofectamine reagent is diluted into
100 .mu.l of Opti-MEM. The two solutions are combined, mixed
gently, and incubated at room temperature for 45 minutes to allow
DNA-liposome complexes to form. For each transfection, 0.8 ml of
Opti-MEM is added to the tube containing the complexes. The diluted
solution is mixed gently and overlaid onto the rinsed cells. The
cells are incubated with the complexed DNA/Lipofectamine for 6
hours at 37.degree. C. in a CO.sub.2 incubator. Following
incubation, 1 ml of growth medium (high glucose MEM) with 20% FBS
is added to each well and incubated at 37.degree. C. overnight. The
medium is replaced with fresh, complete medium at 18 hours
following the start of transfection, and the cells are left to grow
at 37.degree. C. for another 48 hours.
[0084] B. Tau Phosphorylation Inhibition Assay
[0085] Two hours before harvesting, 2 .mu.l of GSK3 inhibitor
dissolved in DMSO is added to each well and incubated at 37.degree.
C. After 2 hours the medium is removed and the cells are rapidly
frozen on the plates on dry ice and stored at -70.degree. C. Cells
are thawed on ice in the presence of 200 .mu.l of lysing buffer (1%
Triton.RTM. X-100, 20 mM Tris pH 7.5, 137 mM NaCl, 15% glycerol, 25
.mu.g/ml leupeptin, 1 .mu.g ml pepstatin-A, 1 PM PMSF, 21 .mu.g/ml
aprotinin, 50 mM NaF, 50 mM .beta.-glycerophosphate, 15 mM sodium
pyrophosphate, 1 mM sodium orthovanadate). The contents of each
well are centrifuged at 14,000 g, 4.degree. C. for 5 minutes and
the supernatants transferred to clean tubes. At this point the
lysates may be stored at -20.degree. C.
[0086] C. ELISA to Detect Phosphorylated Tau in Cell Lysates
[0087] Immulon 4 strips (Dynatech) are coated with monoclonal
anti-phosphorylated tau (AT8, Polymedco, Inc.) at 5 .mu.g/ml in PBS
containing Ca++ and Mg++, 100 .mu.l/well. After overnight
incubation at 4.degree. C., the strips are washed twice with
washing buffer (PBS containing 0.05% Tween.RTM. 20) and blocked
with PBS containing 1% BSA, 5% normal mouse serum and 0.05%
Tween.RTM. 20 at room temperature for 1 hour. The strips are washed
5 times with washing buffer. Lysate (100 .mu.l) diluted 1:10 in PBS
containing 1% BSA, 0.1% NaN.sub.3 is added into each well and
incubated at room temperature for 1 hour. After washing, 100 .mu.l
of 0.5 .mu.g/ml biotinylated monoclonal anti-(non-phosphorylated)
tau (HT7, Polymedco, Inc.) in PBS-BSA is added into each well.
Strips are washed 5 times and HRP-conjugated streptavidin is added,
incubated at room temperature for 30 minutes and washed extensively
with washing buffer. TMB substrate (Pierce) is used for color
development and the reaction is stopped by adding an equal volume
of 0.8 M sulfuric acid. Strips are read on an ELISA plate reader
using a 450 nm filter. The concentration of compound that inhibits
tau phosphorylation to 50% of the maximal level (i.e., IC.sub.50)
is determined by fitting a sigmoidal curve to the plotted data.
[0088] Those skilled in the art will recognize, or be able to
ascertain, using not more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such specific embodiments and equivalents are intended to
be encompassed by the following claims.
[0089] All patents, published patent applications, and publications
cited herein are incorporated by reference as if set forth fully
herein.
Sequence CWU 1
1
11 1 420 PRT Homo sapiens 1 Met Ser Gly Arg Pro Arg Thr Thr Ser Phe
Ala Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala Phe Gly
Ser Met Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys Val Thr
Thr Val Val Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg Pro Gln
Glu Val Ser Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60 Gly Ser
Phe Gly Val Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65 70 75 80
Leu Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg 85
90 95 Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg
Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu
Val Tyr Leu 115 120 125 Asn Leu Val Leu Asp Tyr Val Pro Glu Thr Val
Tyr Arg Val Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys Gln Thr Leu
Pro Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr Gln Leu Phe
Arg Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile Cys His Arg
Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185 190 Thr Ala
Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val 195 200 205
Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala 210
215 220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp
Val 225 230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu
Gly Gln Pro Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val Asp Gln Leu
Val Glu Ile Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr Arg Glu Gln
Ile Arg Glu Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe Lys Phe Pro
Gln Ile Lys Ala His Pro Trp Thr Lys Val 290 295 300 Phe Arg Pro Arg
Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Arg Leu 305 310 315 320 Leu
Glu Tyr Thr Pro Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala 325 330
335 His Ser Phe Phe Asp Glu Leu Arg Asp Pro Asn Val Lys His Pro Asn
340 345 350 Gly Arg Asp Thr Pro Ala Leu Phe Asn Phe Thr Thr Gln Glu
Leu Ser 355 360 365 Ser Asn Pro Pro Leu Ala Thr Ile Leu Ile Pro Pro
His Ala Arg Ile 370 375 380 Gln Ala Ala Ala Ser Thr Pro Thr Asn Ala
Thr Ala Ala Ser Asp Ala 385 390 395 400 Asn Thr Gly Asp Arg Gly Gln
Thr Asn Asn Ala Ala Ser Ala Ser Ala 405 410 415 Ser Asn Ser Thr 420
2 394 PRT Homo sapiens 2 Met Glu Tyr Met Pro Met Glu Gly Gly Gly
Met Ser Gly Arg Pro Arg 1 5 10 15 Thr Thr Ser Phe Ala Glu Ser Cys
Lys Pro Val Gln Gln Pro Ser Ala 20 25 30 Phe Gly Ser Met Lys Val
Ser Arg Asp Lys Asp Gly Ser Lys Val Thr 35 40 45 Thr Val Val Ala
Thr Pro Gly Gln Gly Pro Asp Arg Pro Gln Glu Val 50 55 60 Ser Tyr
Thr Asp Thr Lys Val Ile Gly Asn Gly Ser Phe Gly Val Val 65 70 75 80
Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu Leu Val Ala Ile Lys Lys 85
90 95 Val Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu Leu Gln Ile Met
Arg 100 105 110 Lys Leu Asp His Cys Asn Ile Val Arg Leu Arg Tyr Phe
Phe Tyr Ser 115 120 125 Ser Gly Glu Lys Lys Asp Glu Val Tyr Leu Asn
Leu Val Leu Asp Tyr 130 135 140 Val Pro Glu Thr Val Tyr Arg Val Ala
Arg His Tyr Ser Arg Ala Lys 145 150 155 160 Gln Thr Leu Pro Val Ile
Tyr Val Lys Leu Tyr Met Tyr Gln Leu Phe 165 170 175 Arg Ser Leu Ala
Tyr Ile His Ser Phe Gly Ile Cys His Arg Asp Ile 180 185 190 Lys Pro
Gln Asn Leu Leu Leu Asp Pro Asp Thr Ala Val Leu Lys Leu 195 200 205
Cys Asp Phe Gly Ser Ala Lys Gln Leu Val Arg Gly Glu Pro Asn Val 210
215 220 Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe
Gly 225 230 235 240 Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp Ser
Ala Gly Cys Val 245 250 255 Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile
Phe Pro Gly Asp Ser Gly 260 265 270 Val Asp Gln Leu Val Glu Ile Ile
Lys Val Leu Gly Thr Pro Thr Arg 275 280 285 Glu Gln Ile Arg Glu Met
Asn Pro Asn Tyr Thr Glu Phe Lys Phe Pro 290 295 300 Gln Ile Lys Ala
His Pro Trp Thr Lys Val Phe Arg Pro Arg Thr Pro 305 310 315 320 Pro
Glu Ala Ile Ala Leu Cys Ser Arg Leu Leu Glu Tyr Thr Pro Thr 325 330
335 Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala His Ser Phe Phe Asp Glu
340 345 350 Leu Arg Asp Pro Asn Val Lys His Pro Asn Gly Arg Asp Thr
Pro Ala 355 360 365 Leu Phe Asn Phe Thr Thr Gln Glu Leu Ser Ser Asn
Pro Pro Leu Ala 370 375 380 Thr Ile Leu Ile Pro Pro His Ala Arg Ile
385 390 3 361 PRT Homo sapiens 3 Met Glu Tyr Met Pro Met Glu Gly
Gly Gly Gly Ser Lys Val Thr Thr 1 5 10 15 Val Val Ala Thr Pro Gly
Gln Gly Pro Asp Arg Pro Gln Glu Val Ser 20 25 30 Tyr Thr Asp Thr
Lys Val Ile Gly Asn Gly Ser Phe Gly Val Val Tyr 35 40 45 Gln Ala
Lys Leu Cys Asp Ser Gly Glu Leu Val Ala Ile Lys Lys Val 50 55 60
Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu Leu Gln Ile Met Arg Lys 65
70 75 80 Leu Asp His Cys Asn Ile Val Arg Leu Arg Tyr Phe Phe Tyr
Ser Ser 85 90 95 Gly Glu Lys Lys Asp Glu Val Tyr Leu Asn Leu Val
Leu Asp Tyr Val 100 105 110 Pro Glu Thr Val Tyr Arg Val Ala Arg His
Tyr Ser Arg Ala Lys Gln 115 120 125 Thr Leu Pro Val Ile Tyr Val Lys
Leu Tyr Met Tyr Gln Leu Phe Arg 130 135 140 Ser Leu Ala Tyr Ile His
Ser Phe Gly Ile Cys His Arg Asp Ile Lys 145 150 155 160 Pro Gln Asn
Leu Leu Leu Asp Pro Asp Thr Ala Val Leu Lys Leu Cys 165 170 175 Asp
Phe Gly Ser Ala Lys Gln Leu Val Arg Gly Glu Pro Asn Val Ser 180 185
190 Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala
195 200 205 Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp Ser Ala Gly Cys
Val Leu 210 215 220 Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe Pro Gly
Asp Ser Gly Val 225 230 235 240 Asp Gln Leu Val Glu Ile Ile Lys Val
Leu Gly Thr Pro Thr Arg Glu 245 250 255 Gln Ile Arg Glu Met Asn Pro
Asn Tyr Thr Glu Phe Lys Phe Pro Gln 260 265 270 Ile Lys Ala His Pro
Trp Thr Lys Val Phe Arg Pro Arg Thr Pro Pro 275 280 285 Glu Ala Ile
Ala Leu Cys Ser Arg Leu Leu Glu Tyr Thr Pro Thr Ala 290 295 300 Arg
Leu Thr Pro Leu Glu Ala Cys Ala His Ser Phe Phe Asp Glu Leu 305 310
315 320 Arg Asp Pro Asn Val Lys His Pro Asn Gly Arg Asp Thr Pro Ala
Leu 325 330 335 Phe Asn Phe Thr Thr Gln Glu Leu Ser Ser Asn Pro Pro
Leu Ala Thr 340 345 350 Ile Leu Ile Pro Pro His Ala Arg Ile 355 360
4 483 PRT Homo sapiens 4 Met Ser Gly Gly Gly Pro Ser Gly Gly Gly
Pro Gly Gly Ser Gly Arg 1 5 10 15 Ala Arg Thr Ser Ser Phe Ala Glu
Pro Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Pro Gly Gly
Ser Ala Ser Gly Pro Gly Gly Thr Gly Gly 35 40 45 Gly Lys Ala Ser
Val Gly Ala Met Gly Gly Gly Val Gly Ala Ser Ser 50 55 60 Ser Gly
Gly Gly Pro Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro 65 70 75 80
Gly Ala Gly Thr Ser Phe Pro Pro Pro Gly Val Lys Leu Gly Arg Asp 85
90 95 Ser Gly Lys Val Thr Thr Val Val Ala Thr Leu Gly Gln Gly Pro
Glu 100 105 110 Arg Ser Gln Glu Val Ala Tyr Thr Asp Ile Lys Val Ile
Gly Asn Gly 115 120 125 Ser Phe Gly Val Val Tyr Gln Ala Arg Leu Ala
Glu Thr Arg Glu Leu 130 135 140 Val Ala Ile Lys Lys Val Leu Gln Asp
Lys Arg Phe Lys Asn Arg Glu 145 150 155 160 Leu Gln Ile Met Arg Lys
Leu Asp His Cys Asn Ile Val Arg Leu Arg 165 170 175 Tyr Phe Phe Tyr
Ser Ser Gly Glu Lys Lys Asp Glu Leu Tyr Leu Asn 180 185 190 Leu Val
Leu Glu Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg His 195 200 205
Phe Thr Lys Ala Lys Leu Thr Ile Pro Ile Leu Tyr Val Lys Val Tyr 210
215 220 Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Gln Gly
Val 225 230 235 240 Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val
Asp Pro Asp Thr 245 250 255 Ala Val Leu Lys Leu Cys Asp Phe Gly Ser
Ala Lys Gln Leu Val Arg 260 265 270 Gly Glu Pro Asn Val Ser Tyr Ile
Cys Ser Arg Tyr Tyr Arg Ala Pro 275 280 285 Glu Leu Ile Phe Gly Ala
Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp 290 295 300 Ser Ala Gly Cys
Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe 305 310 315 320 Pro
Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu 325 330
335 Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr Thr
340 345 350 Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys
Val Phe 355 360 365 Lys Ser Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys
Ser Ser Leu Leu 370 375 380 Glu Tyr Thr Pro Ser Ser Arg Leu Ser Pro
Leu Glu Ala Cys Ala His 385 390 395 400 Ser Phe Phe Asp Glu Leu Arg
Cys Leu Gly Thr Gln Leu Pro Asn Asn 405 410 415 Arg Pro Leu Pro Pro
Leu Phe Asn Phe Ser Ala Gly Glu Leu Ser Ile 420 425 430 Gln Pro Ser
Leu Asn Ala Ile Leu Ile Pro Pro His Leu Arg Ser Pro 435 440 445 Ala
Gly Thr Thr Thr Leu Thr Pro Ser Ser Gln Ala Leu Thr Glu Thr 450 455
460 Pro Thr Ser Ser Asp Trp Gln Ser Thr Asp Ala Thr Pro Thr Leu Thr
465 470 475 480 Asn Ser Ser 5 447 PRT Homo sapiens 5 Met Ser Gly
Gly Gly Pro Ser Gly Gly Gly Pro Gly Gly Ser Gly Arg 1 5 10 15 Ala
Arg Thr Ser Ser Phe Ala Glu Pro Gly Gly Gly Gly Gly Gly Gly 20 25
30 Gly Gly Gly Pro Gly Gly Ser Ala Ser Gly Pro Gly Gly Thr Gly Gly
35 40 45 Gly Lys Ala Ser Val Gly Ala Met Gly Gly Gly Val Gly Ala
Ser Ser 50 55 60 Ser Gly Gly Gly Pro Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Pro 65 70 75 80 Gly Ala Gly Thr Ser Phe Pro Pro Pro Gly
Val Lys Leu Gly Arg Asp 85 90 95 Ser Gly Lys Val Thr Thr Val Val
Ala Thr Leu Gly Gln Gly Pro Glu 100 105 110 Arg Ser Gln Glu Val Ala
Tyr Thr Asp Ile Lys Val Ile Gly Asn Gly 115 120 125 Ser Phe Gly Val
Val Tyr Gln Ala Arg Leu Ala Glu Thr Arg Glu Leu 130 135 140 Val Ala
Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu 145 150 155
160 Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu Arg
165 170 175 Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Leu Tyr
Leu Asn 180 185 190 Leu Val Leu Glu Tyr Val Pro Glu Thr Val Tyr Arg
Val Ala Arg His 195 200 205 Phe Thr Lys Ala Lys Leu Thr Ile Pro Ile
Leu Tyr Val Lys Val Tyr 210 215 220 Met Tyr Gln Leu Phe Arg Ser Leu
Ala Tyr Ile His Ser Gln Gly Val 225 230 235 240 Cys His Arg Asp Ile
Lys Pro Gln Asn Leu Leu Val Asp Pro Asp Thr 245 250 255 Ala Val Leu
Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val Arg 260 265 270 Gly
Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro 275 280
285 Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp
290 295 300 Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro
Ile Phe 305 310 315 320 Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu
Ile Ile Lys Val Leu 325 330 335 Gly Thr Pro Thr Arg Glu Gln Ile Arg
Glu Met Asn Pro Asn Tyr Thr 340 345 350 Glu Phe Lys Phe Pro Gln Ile
Lys Ala His Pro Trp Thr Lys Val Phe 355 360 365 Lys Ser Arg Thr Pro
Pro Glu Ala Ile Ala Leu Cys Ser Ser Leu Leu 370 375 380 Glu Tyr Thr
Pro Ser Ser Arg Leu Ser Pro Leu Glu Ala Cys Ala His 385 390 395 400
Ser Phe Phe Asp Glu Leu Arg Cys Leu Gly Thr Gln Leu Pro Asn Asn 405
410 415 Arg Pro Leu Pro Pro Leu Phe Asn Phe Ser Ala Gly Glu Leu Ser
Ile 420 425 430 Gln Pro Ser Leu Asn Ala Ile Leu Ile Pro Pro His Leu
Arg Ser 435 440 445 6 387 PRT Homo sapiens 6 Ser Gly Lys Val Thr
Thr Val Val Ala Thr Leu Gly Gln Gly Pro Glu 1 5 10 15 Arg Ser Gln
Glu Val Ala Tyr Thr Asp Ile Lys Val Ile Gly Asn Gly 20 25 30 Ser
Phe Gly Val Val Tyr Gln Ala Arg Leu Ala Glu Thr Arg Glu Leu 35 40
45 Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu
50 55 60 Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg
Leu Arg 65 70 75 80 Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu
Leu Tyr Leu Asn 85 90 95 Leu Val Leu Glu Tyr Val Pro Glu Thr Val
Tyr Arg Val Ala Arg His 100 105 110 Phe Thr Lys Ala Lys Leu Thr Ile
Pro Ile Leu Tyr Val Lys Val Tyr 115 120 125 Met Tyr Gln Leu Phe Arg
Ser Leu Ala Tyr Ile His Ser Gln Gly Val 130 135 140 Cys His Arg Asp
Ile Lys Pro Gln Asn Leu Leu Val Asp Pro Asp Thr 145 150 155 160 Ala
Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val Arg 165 170
175 Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro
180 185 190 Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp
Val Trp 195 200 205 Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly
Gln Pro Ile Phe 210 215 220 Pro Gly Asp Ser Gly Val Asp Gln Leu Val
Glu Ile Ile Lys Val Leu 225 230 235 240 Gly Thr Pro Thr Arg Glu Gln
Ile Arg Glu Met Asn Pro Asn Tyr Thr 245 250 255 Glu Phe Lys Phe Pro
Gln Ile Lys Ala His Pro Trp Thr Lys Val Phe 260 265 270 Lys Ser Arg
Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Ser Leu Leu 275 280 285 Glu
Tyr Thr Pro Ser Ser Arg Leu Ser Pro Leu Glu Ala Cys Ala His 290 295
300 Ser Phe Phe Asp Glu Leu Arg Cys Leu Gly Thr Gln Leu Pro Asn Asn
305 310 315 320 Arg Pro Leu Pro Pro Leu Phe Asn Phe Ser Ala Gly Glu
Leu Ser Ile
325 330 335 Gln Pro Ser Leu Asn Ala Ile Leu Ile Pro Pro His Leu Arg
Ser Pro 340 345 350 Ala Gly Thr Thr Thr Leu Thr Pro Ser Ser Gln Ala
Leu Thr Glu Thr 355 360 365 Pro Thr Ser Ser Asp Trp Gln Ser Thr Asp
Ala Thr Pro Thr Leu Thr 370 375 380 Asn Ser Ser 385 7 351 PRT Homo
sapiens 7 Ser Gly Lys Val Thr Thr Val Val Ala Thr Leu Gly Gln Gly
Pro Glu 1 5 10 15 Arg Ser Gln Glu Val Ala Tyr Thr Asp Ile Lys Val
Ile Gly Asn Gly 20 25 30 Ser Phe Gly Val Val Tyr Gln Ala Arg Leu
Ala Glu Thr Arg Glu Leu 35 40 45 Val Ala Ile Lys Lys Val Leu Gln
Asp Lys Arg Phe Lys Asn Arg Glu 50 55 60 Leu Gln Ile Met Arg Lys
Leu Asp His Cys Asn Ile Val Arg Leu Arg 65 70 75 80 Tyr Phe Phe Tyr
Ser Ser Gly Glu Lys Lys Asp Glu Leu Tyr Leu Asn 85 90 95 Leu Val
Leu Glu Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg His 100 105 110
Phe Thr Lys Ala Lys Leu Thr Ile Pro Ile Leu Tyr Val Lys Val Tyr 115
120 125 Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Gln Gly
Val 130 135 140 Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val Asp
Pro Asp Thr 145 150 155 160 Ala Val Leu Lys Leu Cys Asp Phe Gly Ser
Ala Lys Gln Leu Val Arg 165 170 175 Gly Glu Pro Asn Val Ser Tyr Ile
Cys Ser Arg Tyr Tyr Arg Ala Pro 180 185 190 Glu Leu Ile Phe Gly Ala
Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp 195 200 205 Ser Ala Gly Cys
Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe 210 215 220 Pro Gly
Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu 225 230 235
240 Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr Thr
245 250 255 Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys
Val Phe 260 265 270 Lys Ser Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys
Ser Ser Leu Leu 275 280 285 Glu Tyr Thr Pro Ser Ser Arg Leu Ser Pro
Leu Glu Ala Cys Ala His 290 295 300 Ser Phe Phe Asp Glu Leu Arg Cys
Leu Gly Thr Gln Leu Pro Asn Asn 305 310 315 320 Arg Pro Leu Pro Pro
Leu Phe Asn Phe Ser Ala Gly Glu Leu Ser Ile 325 330 335 Gln Pro Ser
Leu Asn Ala Ile Leu Ile Pro Pro His Leu Arg Ser 340 345 350 8 15
PRT Artificial Sequence N-terminus addition sequence 8 Glu Phe Met
Pro Thr Glu Ala Met Ala Ala Pro Lys Arg Val Ile 1 5 10 15 9 9 PRT
Artificial Sequence N-terminus addition sequence 9 Glu Tyr Met Pro
Met Glu Gly Gly Gly 1 5 10 6 PRT Artificial Sequence elution
peptide 10 Glu Tyr Met Pro Thr Asp 1 5 11 5 PRT Artificial Sequence
Peptide substrate phosphorylatable by GSK3 11 Ser Xaa Xaa Xaa Ser 1
5
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